Anti-Oxidant and Anti-Inflammatory Activities of Leaves of Barringtonia Racemosa

Full Length Research Paper

Anti-oxidant and anti-inflammatory activities of leaves of Barringtonia racemosa

Mandana Behbahani, Abdul Manaf Ali, Radzali Muse and Noorjahan Banu Mohd

Department of Cell and Molecular Biology, UPM, Malaysia.

Accepted 17, July 2007

The medicinal plant of Barringtonia racemosa (lecythidaceae family) has been used widely in traditional medicine for anti-inflammation and anticancer in Malaysia. The present investigation was carried out to study of the anti-oxidant and anti-inflammatory effects of fully expanded leaf extracts of B. racemosa. Antioxidant activity was measured by using FTC, TBA and DPPH free radical scavenging methods and Griess assay was also used for the measurement of nitric oxide inhibition in lipopolysaccharide (LPS) and interferon- (IFN- )-treated RAW 264.7 cells. Different crude extracts of fully expanded leaf extracts of B. racemosa have exhibited a very good level of nitric oxide (NO) inhibitory and antioxidant activities. In the Griess assay, non polar extracts such as chloroform and hexane extracts were found to be strong inhibitors of NO at different concentrations (25, 50, 100 and 200 µg/ml) in comparison with polar extract (ethanol extract). Chloroform extract didn’t show cytotoxicity against RAW 264.7 cells at different concentrations contrary to hexane and ethanol extracts. The chloroform and hexane extracts exhibited very strong antioxidant properties when compared to Vitamin E (a-tocopherol) in the FTC and TBA methods, the chloroform and hexane extracts exhibited the radical scavenging activity with an IC50 value of 54.29 and 63 µg/ml respectively. Results demonstrated that chloroform extract of B. racemosa leaf may have the potential to be used as anti-inflammation and anti-oxidant agents and it was revealed that the active compound in B. racemosa is lycopene.

Key words: Barringtonia racemosa, anti-oxidant, anti-inflammatory.

INTRODUCTION

Barringtonia racemosa is a tropical higher plant and is a has anti-tumor property and toxicity in mice (Khan et al., member of the Lecythidaceae family. B. racemosa is a 2001). whilst the ethanol extract of the plant leaves dis- moderate sized evergreen tree found in the West Coast played cytotoxicity against the HelA (human cervila of India, Sundarbans, Assam and Andaman Islands and carcinoma) cell line with a IC50 value of 10 g/ml Malaysia. It is used as a traditional medicine in Malaysia (Mackeen et al., 1997).The aqueous extract isolated from and locally known as putat. Its fruits are used to treat the stem bark of B. racemosa has been displayed to have cough, asthma and diarrhea; the seeds are aromatic and antinociceptive and toxicological effect on rats (Thomas useful in treating colics and ophthalmic problems. Pre- et al., 2002) while an ethanol extract of the roots of B. vious studies on some Barringtonia species including racemosa provided two novel clerodane diterpenoid Barringtonia asiatica, Barringtonia acutangul and Barring- nasimalun A and B by NMR and MS analysis( Khan et tonia lanceolata have showed that most of the species al., 2000). possess medicinal properties (Grosvenor et al., 1995; B. racemosa well known in Malaysia as a traditional Khan and Omoloso, 2002). Other related genus of Bar- medicine has been shown to exhibit anti-oxidant and anti- ringtonia edulis also was reported to possess definitive inflammatory effects. In the recent past, there has been sterility and used to induce abortion (Bourdy and Waller, an increase in the use of plants as sources of natural 1992). anti-oxidants for the scavenging of free radicals. The Previous investigation on the bioactivity of B. racemosa latter are known to initiate a series of (oxygen robbing) has shown that the ethanol extract of B. racemosa barks chain reactions resulting in oxidative tissue damage and a wide range of degenerative diseases, such as cancer and a host of cardiovascular diseases (Galati and O'Brien, 2004). *Corresponding Author email: [email protected] Deraniyagala et al. (2003) reported that the aqueous 096 J. Med. Plant. Res.

Table 1. Percentage of antioxidant activity of leaves of Barringtonia racemosa extracted with three different solvents (chloroform, ethanol and hexane).

Antioxidant activity of Barringtonia racemosa (%) Tocopherolic extract Chloroform extract Hexane extract Ethanol extract FTC TBA FTC TBA FTC TBA FTC TBA 67.08±1.32 46.4±1.54 79.13±1.75 58±1.42 76.52±1.64 54.4±1.14 51,39±0.30 33±0.54

Derived leaves were separately extracted using chloroform, hexane and ethanol at room temperature. Each value represents the mean ± SD (n = 3).

bark extract of B. racemosa have several bioactivities interferon – ( Heras et al., 2001). such as antinociceptive effect and this effect was media- ted through opioid mechanisms. Culture of RAW 264.7 Cell The literature survey revealed that there are no scientific studies carried out regarding antioxidant and The murine monocytic macrophage cell line RAW 264.7 was anti-inflammatory activity of the leaves of B. racemosa. purchased from American Type Culture Collection (ATCC, USA). Hence, the present study is focused to evaluate the This macrophase cell line was initially established from a tumor induced by Abelson murine leukemia virus in Balb/c mice (Raschke antioxidant and anti-inflammatory potentials of different et al., 1978). The RAW 264.7 cells were cultured in Dulbecco’s extracts of leaves of B. racemosa. Modified Eagle Media (DMEM) (containing 4 mM L-glutamine, 4.5 g/L glucose, 1.5 g/L (w/v) sodium bicarbonate, 50 U/ml (w/v) penicillin, 50 Ag/ml (w/v) streptomycin) with 10% foetal calf serum MATERIAL AND METHOD (FCS). The cells were cultured at 37°C with 5% CO2 and were split twice a week. The RAW 264.7 cells were semi-adherent cells, Plant material where some cells grew in suspension, some were loosely attached

to the surface and others flatted out and attached to the flask. The Ten kg of the fresh fully expended leaves of B. racemosa were subcultures were prepared by scraping and the sub cultivation ratio collected from Herbal unit, at University Putra Malaysia, in was between 1:3 to 1:6 (culture: media). December 2005. Young leaves (10 – 15 cm long) as well as older leaves (15 – 25 cm long) were chosen. The petioles were removed. The leaves without petioles were washed under running water, air Griess assay for nitric oxide inhibitory activity dried and ground. Out of 10 kg of the fresh leaves of B. racemosa, 1.8 kg of the dried powder was obtained. The dried plants were Briefly, cells were seeded in 96-well tissue culture plates (1 × 106 stored in plastic bags and kept in a dark at room temperature (28 – cells/100 µl) and incubated for 24 h at 37ºC with 5% CO2. One 30°C). hundred µl of chloroform, hexane and ethanol extracts in 0.1% DMSO was then added separately and serially diluted to give final concentrations of 25, 50, 100 and 200 µg/ml (Table 1). Cells were Extraction of compound using different organic solvents then stimulated with 200 U/ml (w/v) of IFN-g and 10 µg/ml (w/v)

LPS for another 17 h. The presence of nitrite, a stable oxidized The dried plant sample (60 g) was placed in a stopped conical flask product of NO, was determined in cell culture media by Griess and macerated with 500 ml of 98% chloroform (Merck, Germany) at reagent (1%, w/v) of sulfanamide and 0.1% (w/v) N-(1-naphtyl) room temperature (28 – 30°C) for 3 days with occasional stirring. ethylenediamine dihydrochloride in 2.5% (v/v, H PO ). One hundred This experiment has done three times. The solvent was then filtered 3 4 µl of cell culture supernatant was removed and combined with 100 and evaporated in a rotary evaporator (Somatco, Germany) under µl of Griess reagent in a 96-well plate followed by spectro- vacuum at 45°C. The residue was placed in oven (Napco, USA) at photometric measurement at 550 nm using a microplate reader. 40ºC until dry. The crude extract was stored in a well-closed Nitrite concentration in the supernatants was determined by container and protected from light and kept in a refrigerator at −4°C. comparison with a sodium nitrite standard curve. The amount of live The extraction was carried out on three separate occasions (n = 3). cells was detected by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl- The above procedure was repeated using 95% ethanol and 98% tetrazolium bromide (MTT) cytotoxicity assay and values given in hexane(Merck, Germany) as solvent instead of chloroform, again percent cell cytotoxicity. replicated on three occasions (n = 3). Out of 60 g of the dried plant sample of B. racemosa could be able to produce 15 g ethanol, 14 g hexane, 18 g chloroform extracts. Measurement of nitrite

For determination of nitric oxide concentration, the stable conver- Anti-inflammatory activity sion product of nitric oxide, nitrite (NO2−) was measured using the Griess reagent (Chi et al., 2001). After 24 h incubation 50µl of Inflammation is characterized by the over production of nitric supernatant from each well of cell culture plates was transferred oxidide (NO). The inducible isoform of NO synthase (iNOS) is into 96-well microplates and equal volume of Griess reagent (1% directly responsible for the generation of NO. Selective inhibition of sulfanilamide, 0.1% (w/v) of N-(1-naphthyl)-ethyline diamine iNOS and its biosynthetic product NO, has been shown to suppress hydrochloride, 2.5% (v/v, H3PO4) was then added to the super- inflammation in a variety of inflammation states. Griess assay has natant at room temperature. The absorbance at 550 nm was been used here to determine the inhibition of nitric oxid formation determined using a Spectramax Plus (Molecular Devices) UV-Vis in RAW 264.7 cells induced by lipo polysaccharide (LPS) and microplate reader after 10 min. The concentrations of nitrite were Behbahani et. al. 097

derived from regression analysis using serial dilutions of sodium w/v), which is as the antioxidant. The concentration of standard nitrite as a standard. Percentage inhibition was calculated based on solution of lycopene was 1 mg/ml. the ability of extracts to inhibit nitric oxide formation by cells The concentration of chloroform extract of B. racemosa was also compared with the control (cells in media without extracts 1 mg/ml. All solutions were stored in brown flasks at −20°C. containing triggering agents and DMSO), which was considered as 0% inhibition. Analysis of lycopene by high performance liquid chromatography Ferric thiocyanate (FTC) method Determination of lycopene was done on chloroform extract of B. The ferric thiocyanate was performed by using Osawa and Namiki racemosa leaves by the modified method of Xu et al. (2006). method (1981). FTC method was used to determine the amount of Isocratic HPLC analysis was performed using a Jasco Series peroxide at the initial stage of lipid peroxidation. The peroxide HPLC (Jasco, USA) system equipped with 1580 pump and a UV- reacts with ferrous chloride (FeCl2) to form a reddish ferric chloride 1570 detector. Peaks were separated on a Diamonsil C18 column (FeCl3) pigment. In this method, the concentration of peroxide using the mobile phase such as acetonitrile, dichloromethane decreases as the antioxidant activity increases. A mixture of 4 mg (25:75, v/v). of sample was placed in 4 ml of absolute ethanol (Merck, To 250 ml of dichloromethane was mixed with 750 ml of Germany), 4.1 ml of 2.52 % (v/v) of linoleic acid (Sigma, USA) in acetonitrile. The mobile phase solution was sieved through 0.45 µm absolute ethanol, 8 ml of 0.05 M phosphate buffer (pH 7.0) and 3.9 Millipore (Millipore, USA) filter then degassed in an ultra sonicator ml of water was placed in a vial with a screw cap and then placed in (Sonicator, USA) for 25 min (Xu et al., 2006) to prevent from the an oven at 40°C in the dark. bubbles. To 0.1 ml of this solution, 9.7 ml of 75% (v/v) of ethanol and 0.1 The flow-rate of the mobile phase was 1.5 ml/min. The ml 30% (w/v) of ammonium thiocyanate (Sigma, USA) was added. absorption of analyses was done at 450 nm. Samples were injected Exactly 3 min after the addition of 0.1 ml of 0.02 M ferrous chloride manually, and the injection volume was 10 l. In this case, T 2000 in 3.5% (v/v) of hydrochloric acid (HCl) to the reaction mixture. The software was used for peak integration and calculation. absorbance was measured at 500 nm every 48 h until the absor- To identify the peaks, the spectral patterns and retention time of bance of the control reached maximum. the samples were compared with standard (lycopene). The control and standard were subjected to the same procedures as the sample, except that for the control, only the solvent was added, and for the standard, 4 mg of sample was replaced with 4 Statistical analysis mg of vitamin E (Endrini et al., 2002). Each experiment was performed in triplicate and repeated two times. The experiments were performed using complete DPPH free radical scavenging activity randomized design (CRD) design and results were analyzed using

The method of DPPH free radical scavenging was measured based one way ANOVA. Experiment was a factorial. Statistical analysis on a method of Yen and Hsieh (1997). The reaction mixture of 1 ml was performed by using Software SAS, Version 6.12. Probability P of 0.3 mM diphenyl-p-picrylhydrazyl radical (DPPH) in ethanol and < 0.05 were considered significant.

0.5 ml of different concentrations of leaf extracts of B. racemosa (500, 250, 125, 63, 32, 16 and 8 µg/ml) was left in the dark at room RESULTS temperature for 30 min. The remaining DPPH was measured by colorimetry at 517 nm. This method was used to determine DPPH Antioxidant activity free radical scavenging activity of compound isolated from B. racemosa leaves Ferric thiocyanate method

Thiobarbituric acid (TBA) method Figure 1 shows absorbance values obtained using FTC method, for B. racemosa leaves extracted using absolute The method of Ottolenghi (1959) was used to determine the TBA ethanol, chloroform and hexane. Absorbance values of values of the samples. The formation of malonaldehyde is the basis for the well-known TBA method used for evaluating the extent of the control as well as -tocopherol and Baringtonia lipid peroxidation. At low pH and high temperature (100°C), leaves increased until day 6, and then decreased on day malonaldehyde binds TBA to form a red complex that can be 8. In comparing the total antioxidant activity of leave measured at 532 nm. The increase amount of the red pigment extracted using 3 different solvents with vitamin E (- formed correlates with the oxidative rancidity of the lipid. tocopherol), the result showed that nonpolar extract One ml sample solution was prepared and incubated as in the FTC method. 2 ml of 20% (w/v) of trichloroacetic acid (CCl3COOH) and (chloroform and hexane extracts) was significantly higher 2 ml TBA aqueous solution were added to 1 ml of sample solution (p < 0.05) in total antioxidant activity content than - prepared as in the FTC procedure, and incubated in a similar tocopherol. The chloroform extract of B. racemosa had manner. The mixture was then placed in a boiling water bath for 10 the highest activity (79.13 ± 1.75%), followed by hexane min. After cooling, it was centrifuged at 3,000 rpm for 20 min and extract (76.52 ± 1.64%), tocopherol (76.52 ± 1.64%) and the absorbance of the supernatant was measured at 532 nm. ethanol extract (51.39 ± 0.30%) by using FTC method. Antioxidant activity was determined based on the absorbance on the final day. The FTC method was used to measure the amount of peroxide at the primary stage in linoleic acid peroxidation. The control showed increasing in the absorbance value Sample preparation from day 1 to day 4, but the levels reached maximum on Standard solution was prepared by dissolving pure individual day 6 and finally dropped on day 8 due to the MDA compound including lycopene in chloroform containing BHT (1%, compounds from linoleic acid oxidation, where the pero- 098 J. Med. Plant. Res.

1.2

) 1

m Chloroform extract n

0 0.8 Hexane extract 5 (

c 0.6 Tocopherol n a b

r 0.4 Ethanol extract o s

b Control A 0.2

0 1 4 6 8 Day

Figure 1. Absorbance value of B. racemosa leaf at 0.02% concentration using FTC method. The values are compared with the absorbance values of the control and -tocopherol at 500 nm. Each value represents the mean of three replications ± SD.

)

% 60 ( chloroform extract y t i 50 v

i Hexane extract t c

a 40 Tocopherol t n

a 30 Ethanol extract d i x o

i 20 t n

A 10

0 FTC TBA

Figure 2. The total antioxidant activity of chloroform and Ethanol extracts of B. racemosa by using FTC and TBA method. Values are means ± SD (vertical lines). FTC: Ferric thiocyanate, TBA: Thiobarbituric acid

xide reacts with ferrous chloride to form a reddish ferric method, for B. racemosa using three different solvents chloride pigment. Since the concentration of the peroxide chloroform, hexane and ethanol. The TBA method was decreased as the antioxidant activity increase, the inten- used to measure the secondary stage of linoleic acid sity of the reddish pigment will reduce, leading to lower peroxidation. The absorbance values of the chloroform absorbance value. As such, the absorbance values of the and hexane extracts were lower than the control and - control were the highest since it did not contain antiox- tocopherol, on day 8 of the FTC method due to the idants. formation of MDA. Malonaldehyde binds TBA to form a

red complex that can be measured at 532 nm. As shown Thiobarbituric acid method in Table 1, result obtained from the TBA method showed that total antioxidant activity of chloroform and hexane Figure 2 shows absorbance values obtained using TBA extracts were significantly (p < 0.05) higher compared to Behbahani et. al. 099

90 80

) 70 % (

n 60

o Tocopherol i t

c 50 Chloroformic extract u d

e Hexanic extract

r 40

H Ethanolic extract P 30 P

D 20 10 0 8 16 32 63 125 250 500 Concentration(ug/ml)

Figure 3. Free radical scavenging activity of leaf extracts of Barringtonia racemosa determined by DPPH free radical. Each value represents the mean of three replications ± SD.

Table 2. Anti-inflammatory activities of leaves of Barringtonia DPPH radical scavenging activity racemosa extracted with hexane, chloroform and ethanol. The model of scavenging the stable DPPH radical is a Conc. (µg/ml) NO inhibition (%) cell viability widely used method to evaluate antioxidant activities in a Chloroform extract relatively short time compared with other methods. Figure 25 23.40± 1.5 131 3 shows the free radical scavenging activity of chloro- 50 49.62 ± 2.4 125 form, Hexane and ethanol extracts of B. racemosa and one reference compound (-tocopherol). -tocopherol 100 57.70± 2.3 109 was the most potent scavenger. IC50 values of - 200 73.85 ± 2.5 100 atocopherol, chloroform, hexane and ethanol extracts Ethanol extract were found to be 32, 54, 63 and 125 µg/ml respectively. 25 16.14 ± 2.0 84 IC50 values of plant extracts tested showed lower activity 50 22.65 ± 1.5 93 than -atocopherol. The result showed that non-polar 100 29.80± 2.2 91 extract (chloroform and hexane extracts) was significantly higher (p < 0.05) in DPPH activity compared to polar 200 45.60± 2.1 95 extract (ethanol extract). Among two kind of non polar Hexane extract extracts, chloroform extract was significantly higher (p < 25 25.80± 3.1 111 0.05) in DPPH activity compared to hexane extract. 50 37.79 ± 2.9 85

100 42.39 ± 2.0 55 The Effect of different leave extracts on NO 200 60.60± 1.6 41 production in RAW 264.7 Cells

The overall efficacy of all extracts on nitrite production in IFN /LPS activated macrophage is shown in Table 2. vitamin E. Among nonpolar and polar solvents, there was Hexane, chloroform and ethanol extracts of B. significantly difference (p < 0.05) in total antioxidant raceomosa were examined for their inhibitory effect on activity. As in FTC method, the lower absorbance value NO release in RAW264.7 cells. Each sample was as- indicated higher antioxidant activity. Since peroxide sayed at concentration of 25, 50, 100 and 200 µg/ml with production was inhibited by the antioxidant, similarly to 0.2% DMSO. All samples were compared with control; the FTC method findings, chloroform extract showed to the non-treated LPS activated macrophage in culture have the highest content of total antioxidants activity media. The extracts were considered as having a strong, compared to hexane and ethanol extracts. moderate, or week activity if inhibition of NO release was 100 J. Med. Plant. Res.

Figure 4. HPLC chromatogram of fully expanded leaf extract of Barringtonia racemosa spectra detected by UV-1570 detector at 450 nm using acetonitrile: dichloromethane (25: 75, v/v) solvent system at the flow rate of 1 ml/min. A, C, E, G, I, J and H indicate the unknown peaks. F indicate standard lycopene peak.

was more than 90%, between 50 and 90%,or less than HPLC chromatogram of lycopene and leaf extract of 50% compared to the induced (with 0.1% DMSO) B. racemosa treatment, respectively. In addition, the viability of RAW 264.7 cells was assessed by MTT method (Mossman, The result showed that the retention time of standard 1983) and must be above 85% indicating that decreased lycopene was 6.28. The leaf extract of B. racemosa NO were not a result of cell death but rather due to the showed ten absorption peaks at 450 nm (Figure 4). The inhibitory effect of the compound, where applicable. peak areas of lycopene are shown in Figure 5 (F). Among the tree extracts (Table 2), only chloroform Repeated experiments showed that, the retention time of extract showed higher than 50% inhibition of NO lycopene and leaf extract of B. racemosa were almost the production at the concentration of 200 and 100 mg/ml same. The other peaks that observed in fully expanded without cytotoxic effect. On the other hand, chloroform leaf extract of B. racemosa as shown in Figure 4 (A, B, C, extract showed an enhanced effect rather than an D, E, I, J and H ) were unknown compounds. inhibitory effect on percentage of cell viability. Hexane extract showed higher than 50% inhibition of NO DISCUSSION AND CONCLUSION production at the highest concentration 200 µg. This extract did not indicate cytotoxicity at concentration 25 Results obtained from this study revealed that the active µg/ml in cultures as determined by MTT assay but was compound in B. racemosa is lycopene. Lycopene is a highly cytotoxic at higher concentrations (50, 100 and major antioxidant present in a range of fresh fruits and 200 µg/ml). Ethanol extract showed less than 50% vegetables. This compound is known to take part in inhibition of NO production at different concentrations and protecting animals against damage from free radicals and showed low degree of cytotoxicity against RAW 264.7 singlet oxygen reactive species. In a study with mice, cells. lycopene has been shown to possess excellent anti- As a result only Chloroform extract compared to the inflammatory/antioxidant properties (Zhao et al., 2003). other two extracts didn’t show cytotoxicity against RAW Lycopene has been shown to suppress the growth of 264.7 cells at different concentrations and was found to cancer cells in laboratory and animal studies. Additional be strong inhibitors of NO at different concentrations. It recent studies in humans have bolstered these findings, could be concluded that chloroform is the best Solvent for showing significant benefits from elevated lycopene extraction of active compound from leaves of B. levels both in the diet and in the body (Welsch et al., racemosa. 2001). Behbahani et al. 101

Figure 5. HPLC chromatogram of standard lycopene spectra detected by UV-1570 detector at 450 nm using acetonitrile, dichloromethane (25:75) solvent system at the flow rate of 1 ml/min. F indicates standard lycopene peak.

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